Acinetobacter baumannii causes hospital infections and negatively impacting patient outcome. A. baumannii is characterized by its exceptional ability to acquire multidrug resistance and spread among hospitalized patients. The rate of resistance to carbapenems in A. baumannii has now exceeded 40% in the U.S. Carbapenem- resistant strains are frequently resistant to all other agents except colistin. Infections due to these organisms are treated with colistimethate (prodrug of colistin) alone or in combination with another antimicrobial agent. However, the increased use of colistin has resulted in the emergence of colistin-resistant A. baumannii. Colistin interacts with the lipid A moiety of lipopolysaccharide and causes disorganization of the membrane. Modification of lipid A with phosphoethanolamine has been proposed as playing a key role in colistin resistance in A. baumannii. Findings from laboratory-generated colistin-resistant mutant strains suggest that this modification carries substantial fitness cost and impairs virulence. However, a subset of patients with colistin- resistant A. baumannii suffer prolonged infection and high mortality. This suggests that colistin-resistant clinical strains have additional mechanisms, which enable them to remain virulent and survive in the human hosts. Our collection of paired colistin-susceptible and -resistant clinical strains from same patients provides us with an unparalleled opportunity to define clinically relevant mechanisms of resistance, fitness and virulence, and their implications for the diagnosis and treatment of colistin-resistant A. baumannii. The central hypotheses to be tested are: (1) lipid A modification is the predominant mechanism of resistance to colistin and can be exploited for rapid diagnosis of resistance, (2) fitness and virulence of colistin-resistant A. baumannii is variable and dependent on the specific mechanism of resistance, and (3) combinations of antimicrobial agents have unique activity against colistin-resistant A. baumannii. To test these hypotheses, we plan to conduct the following Specific Aims: (1) Elucidate the lipid A structures associated with colistin resistance, (2) Characterize the fitness and virulence potential of colistin-resistant A. baumannii and the activity of antimicrobial combinations in in vitro and in vivo models, and (3) Define the genomic and proteomic correlates of colistin resistance. In addition to the existing colistin-resistant clinical strains,those collected prospectively from three hospitals with diverse epidemiology will also be studied, enhancing the generalizability of the findings. The study aims to elucidate the mechanisms of colistin resistance in A. baumannii that are relevant to clinical settings using a multifaceted yet integrated approach. The findings will substantially enhance our understanding of the biology of colistin-resistant A. baumannii, and contribute in devising both novel diagnostic and therapeutic approaches to improve the care of severely ill patients affected by this otherwise untreatable organism.